Although current immunosuppression has dramatically improved organ allograft survival, the majority of patients develop impaired graft function and chronic rejection. Therefore, the ultimate goal--to radically improve graft survival--is to induce transplantation tolerance of allografts without the need for continuous drug therapy. It became obvious that tolerance induction requires two phases, namely, initial deletion (apoptosis) of alloreactive T cells and generation of regulatory T (Treg) cells. We have induced tolerance by promoting apoptosis by anti-CD40 Ligand antibody alone or in combination with CTLA4Ig or apoptosis-inducing agent, WP744. In addition, donor cells or donor/recipient histocompatibility proteins may be added to the protocols to promote generation of interleukin (IL)-4- producing T helper (Th2)reg cells to test our hypothesis that tolerance depends upon the expansion of Th2reg cells with IL-4-driven Jak3/Stat5/Stat6 feedback loops. Function of Th2reg cells is positively controlled by Jak/Stat molecules and negatively controlled by SOCS family proteins, including suppressor of cytokine signaling (SOCS)1, SOCS2, SOCS3, SOCS5, cytokine inducible SH-2 containing protein (CIS)-1, and tyrosine phosphatase SH2 containing protein (Shp)-1. We have developed quantitative real-time PCR (QRT-PCR) method to measure SOCS mRNA expression. Our preliminary experiments showed that tolerance correlated with increased expression of SOCS3 mRNA. Therefore, we plan to examine the kinetics of SOCS expression during induction and maintenance of tolerance by QRT-PCR and Western blot (WB) methods. These results will be correlated with tyrosine phosphorylation (by immunoprecipitation [IP]/WB) and DNA binding (by electrophoretic mobility shift assay [EMSA]) of Stat5 and Stat6 during tolerance induction. We postulate that mice over-expressing SOCS3 should be easier to induce tolerance in comparison to normal or SOCS3-deficient mice. We plan to directly link IL-4-induced Stat5 and Stat6 activation with SOCS3 expression: the IL-4-responsive D10 T cell line will be co-transfected with Stat5- or Stat6-driven luciferase reporter gene and different SOCS. Such an approach will directly evaluate whether Stat5 or Stat6 activation is regulated by SOCS3 and/or other SOCS. These experiments will provide the first evidence for the role of SOCS in induction of transplantation tolerance.